In order to assess the function of hepatocyte nuclear factor 4alpha (HNF4alpha) in adult intestine, an intestinal epithelial cell-specific Hnf4alpha-null mouse line was produced using Cre transgenic mice in which the Cre gene is expressed in the intestine under the control of the mouse villin promoter. These mice exhibited slight decrease of polysaccharides and acidic mucopolysaccharides and altered expression of several genes such as mucins, aquaporins, and meprins. Gut-specific Hnf4alpha-null mice exhibited increased susceptibility to dextran sulfate sodium-induced inflammatory bowel disease (IBD), including increased intestinal permeability. These findings show that HNF4alpha may have an important role in protecting against inflammatory bowel disease. Mice lacking expression of the HNF4alpha gene in intestinal epithelial cells were successfully developed. Disruption of both mRNA and protein level of HNF4alpha were observed in intestine of HNF4alpha intestine-specific knockout mice by northern blot and immunohistochemistry. The levels of serum triglyceride, cholesterol, and phospholipids in HNF4alpha intestine-specific knockout mice mice were normal. The levels of polysaccharides and acidic mucopolysaccharides were decreased in the colon of HNF4alpha intestine-specific knockout mice. A marked decreased expression of mucin3, aquaporins1 and 8, and meprin1alpha and 1beta, and increased expression of mucin1 was also detected in the colon of HNF4alpha intestine-specific knockout mice. After dextran sulfate sodium treatment, HNF4alpha intestine-specific knockout mice mice showed significant severe changes, including loss of body weight, colon length, cytokine levels, histological morphology, and intestinal permeability, compared with control mice. In conclusion, HNF4alpha intestine-specific knockout mice showed higher susceptibility to dextran sulfate sodium-induced inflammatory bowel disease as a result of increased permeability of the epithelial barrier. These data indicate HNF4alpha may play an important role in protection against intestinal inflammatory insult.